COL4A5 primarily affects males. Females with a heterozygous state show a diverse phenotypic spectrum ranging from microscopic hematuria to end-stage kidney disease (ESKD) and extrarenal manifestations. In other X-linked diseases, skewed X-inactivation leads to preferential silencing of one X-chromosome and thus can determine the phenotype in females. We aimed to show a correlation between X-inactivation in blood and urine-derived renal cells and clinical phenotype of females with a heterozygous disease-causing variant in COL4A5 compared to healthy controls. A total of 56 females with a heterozygous disease-causing COL4A5 variant and a mean age of 31.6 ± 18.3 SD years were included in this study. A total of 94% had hematuria, 62% proteinuria >200 mg/day, yet only 7% had decreased eGFR. Using human androgen receptor assay X-inactivation was examined in blood cells of all 56 individuals, in urine-derived cells of 27 of these individuals and in all healthy controls. X-inactivation did not correlate with age of first manifestation, proteinuria or eGFR neither in blood, nor in urine. The degree of X-inactivation showed a moderate association with age, especially in urine-derived cells of the patient cohort . Determination of X-inactivation allelity revealed a shift of X-inactivation toward the COL4A5 variant bearing allele. This is the first study examining X-inactivation of urine-derived cells from female individuals with AS. A correlation between phenotype and X-inactivation could not be observed suspecting other genetic modifiers shaping the phenotype in female individuals with AS. The association of X-inactivation with age in urine-derived cells suggests an escape-mechanism inactivating the COL4A5 variant carrying allele in female individuals with AS.X-linked Alport syndrome (AS) caused by hemizygous disease-causing variants in  COL4A5 or by disease-causing variants in COL4A3 or COL4A4 comparable with an autosomal inheritance (Alport syndrome (AS) is a hereditary nephropathy characterized by (microscopic) hematuria, proteinuria, chronic kidney disease potentially progressing to end-stage kidney disease (ESKD), hearing loss, and typical ocular changes , 2. The eritance .Males with X-linked AS show a distinct genotype-phenotype correlation, illustrated by more severe symptoms in individuals with loss-of-function variants and less severe symptoms in individuals with missense variants or in-frame deletions. Additionally, variants located at the 5′ end have been described to present with earlier onset of ESKD and higher risk of extrarenal manifestations , 11.COL4A5 have traditionally been described as healthy carriers even though the penetrance is variable both intrafamilial and interfamilial, showing a broad spectrum of clinical symptoms, varying from mild isolated microscopic hematuria to severe AS  appear to result in a more severe phenotype–similarly to affected males . There were no comorbidities observed in the included individuals. Exclusion criteria were missing written informed consent and absence of above-mentioned inclusion criteria. Recruitment was primarily achieved by acquiring relatives of known male AS individuals with disease-causing COL4A5 variants. Altogether 60 female individuals were recruited fulfilling the inclusion criteria. Four individuals had to be excluded due to indistinguishable X-inactivation analysis , leading to 56 available individuals from 43 unrelated families. In 49 of those individuals, a blood sample from an AS-affected relative was available needed for determining the potential skewing.Inclusion criteria of the study were proof of a heterozygous disease-causing A total of 43/56 patients were recruited as part of the NephroGen cohort of the Institute of Human Genetics at the Klinikum rechts der Isar of the Technical University of Munich, and 2/56 at the Department of Pediatrics I of the University Children’s Hospital in Heidelberg. The remaining 11 patients were recruited at the Department of Pediatric Nephrology of the University Hospital in Essen, Germany. The study was approved by the respective local ethics committee of each contributing center and performed in accordance to the standards of the 2013 Helsinki Declaration. All individuals or their legal guardians gave written and informed consent.Phenotypic information was gathered by studying medical records and interviews. Age of first manifestation was considered as time point of first proof of an abnormal renal phenotype  or diagnosis of an extrarenal manifestation. When 24-h urine collection was not available, urinary protein/creatinine ratio of more than 200 mg/g creatinine was considered as proteinuria. Semi-quantitative urine dipstick measurements with the result “negative” were considered as proteinuria less than 200 mg/day. Serum creatinine values were determined by the treating physician. eGFR was calculated for individuals >14 years with CKD-EPI formula , for indHealthy female control individuals without history of AS in their families were recruited and blood was drawn as well as spontaneous urine collected. Out of 40 healthy control individuals, five were homozygous for the AR locus and therefore not useable for the control cohort of this study. X-inactivation data of the remaining 35 female healthy controls were available for the study.COL4A5 variants and the clinical phenotype of the affected individuals. Variants were adjusted to the RefSeq Sequence NM_033380.3. Frameshift, non-sense and canonical splice site variants as well as a large deletion  were considered loss-of-function variants. For statistical calculation, two variants with small in-frame deletions were grouped with missense variants . Additionally, they were counted as glycine-affecting variants.The severity of variants was calculated based on a classification of variants according to their position in the transcript. Variants were not located in the signal peptide and NC2 domain. Variants were classified according to their domain and the proximity to the 5′-end. Variants in the 5′-end near collagenous domain (c.124-c.2246) were considered “severe,” variants in the 3′-end near collagenous domain (c.2247-c.4386) were considered “moderate” and variants in the NC1 domain  were considered “mild”  10)..10).DNA was automatically extracted from EDTA blood samples with “Chemagic DNA Blood 5 k Kit” using a Chemagen 360  according to the manufacturer’s instructions.Kidney tissue from biopsy samples is the gold standard to obtain renal-derived cells but was seldom available for analysis. Therefore, we compared X-inactivation in tissue-derived and urine-derived cells from individuals without renal disease and achieved comparable (less than 20% difference) results in 75% of individuals.In addition, urine-derived cells were analyzed in five individuals with AS of the study cohort regarding the presence of podocytes and tubular cells. Therefore, podocytes and tubular cells were isolated from urine samples with magnetic cell separation using the “CD10 Microbead Kit human” . Subsequent FACS analysis confirmed the enrichment of CD10 positive cells. DNA was then extracted from these cells with Qiagen’s “DNeasy Blood and Tissue Kit 250” .The expression of CD10 in podocytes and tubular cells has been demonstrated in immunohistochemical stainings . In 80% AR, Xq11-12; HUMARA-Assay) (The X-inactivation status was measured by assessing the DNA methylation based on the difference between the number of trinucleotide repeats at the human androgen receptor locus (A-Assay) .Hpall) can differentiate between active and inactive alleles. A male sample was enclosed in each assay in order to control sufficient Hpall digestion. Subsequent to digestion and amplification of the samples with fluorescence-tagged PCR primers, fragment length analysis of the PCR products was run on an ABI 3130XL genetic analyzer and the GeneMarker software . If amplified DNA fragments exhibited the same size on both alleles, X-inactivation could not be determined.Due to the correlation of X-inactivation with hypermethylation, digestion with methylation sensitive restriction enzymes . These data are referred to as “X-inactivation without allelity” and are displayed in a range from theoretically 50%  to 100% (completely skewed).X-inactivation data was generated and is displayed in two different ways depending on the availability of an affected male relative. In a first step, skewness of X-inactivation was measured with the ratio of inactivation between the two alleles without knowledge which of the alleles carried the COL4A5 variant bearing allele not inactivated at all) allowed determining which of the two alleles in a female individual with a heterozygous disease-causing variant was the one bearing the COL4A5 variant  to 100% . This dataset is referred to as “X-inactivation with allelity.”In families with an available affected male, segregation analysis of the male individual , was used for all statistical tests.Parametric data are presented as mean ± standard deviation and non-parametric data as median . For correlation analyses Spearman’s rho was calculated . When corameters . SPSS® SCOL4A5 with X-inactivation data in peripheral blood cells resp. in urine-derived cells were available for analysis  with first symptoms/abnormal urine analysis detected at a median age of 9.5  years. Most common manifestation was hematuria  followed by proteinuria >200 mg/day (62.3%), whereas mean eGFR was normal. Four of the 56 individuals progressed to ESKD with a mean age of onset at 32.8 ± 5.3 years (mean ± SD). Extrarenal manifestation defined as hearing impairment (5.4%) or ocular manifestation (7.1%) were detected only in a few individuals.The mean age of the included individuals was 31.6 ± 18.3 years .Loss-of-function variants were present in 42.9% of cases, mostly represented by frameshift and canonical splice site variants . IndividComparing the total cohort of 56 individuals with the 27 individuals with additional urine samples, the subcohort of individuals with X-inactivation data from urine-derived cells were slightly older, had less proteinuria, slightly decreased eGFR and did not include individuals with ESKD due to loss of remaining urine excretion or history of kidney transplantation .n = 35) had no history of AS in the family, were all female and had a mean age of 37.1 ± 19.1 years (mean ± SD).Healthy controls (p = 0.011). Individuals with ocular manifestations or hearing impairment predominately carried loss-of-function variants.Median age of first manifestation was significantly lower in individuals with loss-of-function variants compared to individuals with missense variants  . For the controls, the degree of X-inactivation amounted to median 65%  for blood cells and 66%  for urine-derived cells.Median X-inactivation of cases without considering allelity was 65%  in blood cells and 59%  in urine-derived cells (data not shown). Analysis of rho = 0.348, p = 0.075 and rho = 0.551, p = 0.001; COL4A5-dependent X-inactivation allelity, the association between blood and urine-derived cells was even higher  in urine-derived cells of the affected individuals, a significant association with a Spearman’s rho of 0.403 .Correlation analyses of phenotypic characteristics and X-inactivation could not demonstrate a significant association for age of first manifestation, eGFR or proteinuria . There wn = 4) revealed increased X-inactivation without allelity (77% in ESKD vs. 64% in non-ESKD) as well as increased X-inactivation with COL4A5-dependent allelity  and the degree of X-inactivation of the disease-causing allele amounted to 55% in blood cells and 56% in urine-derived cells. This suggests that X-inactivation could serve as an escape-mechanism protecting individuals from the development of severe complications of AS as they get older. Involvement of X-inactivation has been described for heterozygous carriers in X-linked diseases like severe combined immunodeficiency . These associations were stronger than described in the literature for cells from other slowly proliferating tissues (like fat and skin)  . As bloond skin) , 23, uriCOL4A5 in blood cells only , which also led to decreased numbers of individuals with ESKD and extrarenal manifestations. This bias is based on the recruitment process, which mostly included sisters or mothers of pediatric patients. The degree of skewness in blood cells evidently accelerates above the age of 60 years . Thus, aCOL4A5 variant as individuals get older. A relationship between X-inactivation and phenotypic characteristics was not present in blood as well as urine cells. Future studies about X-inactivation in female individuals should focus on older cohorts.In conclusion, our data show an association of X-inactivation and age in urine-derived cells of female individuals with AS, which could present an escape-mechanism to avoid expression of the The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/This study was approved by the respective local ethics committee of each contributing center. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.RG, LK, SJ, BL-D, CH, KR, JC, and KB performed the analysis. RS, MB, BT, VT, NA-E, VN-S, NG, AM, LP, MK, OG, AL, SW, LR, UH, TM, and AB cared for the patients and acquired and provided the clinical data. RG, AB, and JH were responsible for writing and revision of the manuscript. All authors contributed to the article and approved the submitted version.


Trichoderma enzyme treatment, PX (with endo-xylanase) and PCX (with both endo-cellulase and endo-xylanase), were studied in colon cancer cell lines . Both pectins reduced colon cancer cell viability, induced apoptosis, and increased intracellular amounts of reactive oxygen species. Additionally, synergy between pectin and an active form of irinotecan, SN-38, in all aspects mentioned above, was discovered. This drug is a common component of cytotoxic combinations recommended as treatment for colon cancer patients. PX and PCX demonstrated significant anti-inflammatory activity in lipopolysaccharide-stimulated cells. Interaction of apple pectins with galectin-3 and Toll-like Receptor 4 (TLR4) was suggested to be responsible for their anticancer and anti-inflammatory effect. Since PCX was more active than PX in almost all experiments, the role of the enzyme used to obtain the pectin for its biological activity was discussed. It was concluded that co-operation between both enzymes was needed to obtain the molecule of the most beneficial properties. The low molecular mass of PCX together with a high proportion of rhamnogalacturonan I (RG I) regions seemed to be crucial for its superior activity.Pectin constitutes an essential component of dietary fiber. Modified pectins from various sources possess potent anticancer and immunomodulatory activities. In this study, two pectins isolated from apple pomace by  Pectins are acidic heteropolysaccharides that may constitute up to 40% of the dry mass of plant cell walls  and theyExtracted pectin is mainly used in the food industry as gelling, thickening, emulsifying, and stabilizing agents in a variety of foods . RecentlThe main commercial sources of pectins are citrus peels and apple pomace . IndustrIn our previous study , the prop < 0.05 for all concentrations tested). IC50 values of the studied pectins are presented in 50 values recorded for PCX were ca. twice lower than the ones obtained for PX. The SRB assay experiments brought virtually the same results .This prompted us to perform isobolographic analysis to detect the existence of putative synergy between the apple pectins and the anticancer drug. For pure compounds and their combinations, dose and effect data were obtained from the MTT assay. The analysis via CompuSyn software based on the model of Chou and Martin  yielded p < 0.05 as compared to the untreated control) increase of the sub-G1 fraction of HT-29 cells with a concomitant decrease in other fractions, especially G0/G1. Cells in the sub-G1 phase are predominantly dead cells, i.e., necrotic or late apoptotic, while G0/G1 phase represents living mononuclear cells. The addition of 5 nM of SN-38 also increased the share of sub-G1 cells, however, this was to a lesser extent than pectins themselves. Most interestingly, the combination of pectin and SN-38 resulted in a robust increase in the number of HT-29 cells in the sub-G1 phase. This pointed to the mutual enhancement of activity between the anticancer drug and apple pectins. Similar results were obtained when the influence of the studied pectins was investigated in HCT 116 cells and Caco-2 cells . SN-38 applied at 5 nM concentration also slightly increased the population of apoptotic cells. However, the most pronounced effect was observed when the pectins were used in combination with the anticancer drug. The proportion of apoptotic cells raised dramatically. It is worth noticing that in all cases, the factor responsible for reducing the normal cell population was the significant increase of the number of apoptotic cells, while the population of necrotic cells increased only slightly. Similar results were also obtained for HCT 116 and Caco-2 cells  in cells treated by pectins. As shown in Next, the generation of ROS in colon cancer cells in the presence of apple pectins was quantified. The results of the assay in which intracellular ROS were detected with the use of fluorescent probe DCF are presented in Colon cancer cells were pretreated by lipopolysaccharide (at 0.5 μM) for 24 h to induce inflammation. The ability of apple pectins to modulate the cellular inflammation process was investigated in a model system prepared in such a way. As shown in TLR4 plays the role of main cellular receptor for LPS, and its activation leads to the activation of cytokine production. As shown in Since both studied pectins induced apoptosis in colon cancer cells, it was also checked whether they could affect the cellular amount of galectin-3 . Gal-3 possesses many functions. Among others, it is engaged in apoptosis regulation. Investigation of Gal-3 by means of ELISA assay showed that the treatment of HT-29 cells by PX or PCX (at 0.2 mg/mL) resulted in a pronounced drop in the amount of Gal-3 detected in cell lysates . AdditioEscherichia coli was tested in HCT 116 cells (p < 0.05). PCX was less active in this respect. It exerted a slight inhibition on E. coli adherence at the highest concentration used, however, statistical significance was not reached.Since the interaction of all compounds with colonic cells might be modulated by the local microbiota, the ability of the pectins to interfere with the adherence of 16 cells . The adh50 values recorded for PCX in HT-29 and Caco-2 cells were almost twice lower than those for PX. In HCT 116 cells, IC50 values for both pectins were comparable but PCX was still more cytotoxic than PX. Anticancer properties of PX and PCX have been previously studied in adenocarcinoma and melanoma cells [The anticancer activity of enzymatically extracted apple pectins, PX, and PCX, was demonstrated in a panel of colon cancer cell lines. Both studied pectins were cytotoxic to HCT 116, Caco-2, and HT-29 cells. ICma cells . It was ma cells . Pectinsma cells  and ginsma cells , were prma cells ,26. On tma cells  and papaThe cytotoxic properties of the apple pectins towards colon cancer cells were also studied in the presence of a low concentration of the active form of irinotecan (SN-38). The drug was used at the concentration of 5 nM, which was practically non-toxic to none of the studied cell lines. When SN-38 and apple pectins were applied together, the studied compounds exerted a much more pronounced cytotoxic effect on all types of the colon cancer cells. Isobolographic analysis clearly demonstrated the existence of synergy between the apple pectins and SN-38. For PX, the severity of the observed synergistic effect was similar in all three cell lines studied. On the other hand, PCX exhibited more potent synergy with SN-38 in HCT 116 and Caco-2 cells than in HT-29 cells. Therefore, it was concluded that the processes that resulted in synergy were dependent on the type of cancer cells. Together with our previous observation , this is1 phase of the cell cycle (necrotic and late apoptotic cells) as well as the induction of apoptosis . When the binary combination of the apple pectin and SN-38 was applied to colon cancer cells, the enhancement of the effect of pectins both on the cell cycle and apoptosis induction was recorded. Proapoptotic properties of various pectin preparations in colon cancer cells have been previously reported. Pectins obtained from sugar beetroot and sweet potato were found to induce apoptosis in HT-29 cells [2/M phase and induced apoptosis accompanied by the activation of caspase-3 [The observation of the cytotoxic effect of enzymatically extracted apple pectins on colon cancer cells was supplemented by the study of their effect on cell cycle and apoptosis induction. In all three cell lines, the presence of pectins resulted in an increase in the number of cells in the sub-G29 cells ,32. The aspase-3 . Enzymataspase-3 , and enzaspase-3 . Finallyaspase-3 .Campomanesia xanthocarpa [Next, the ability of the apple pectins to affect the level of lipid peroxidation and intracellular ROS was investigated. It was found that the studied pectins significantly increased the levels of both markers of oxidative stress in colon cancer cells. Additionally, in the presence of SN-38, the effect of pectins was highly augmented. This suggested that pectins displayed pro-oxidative properties in the experimental settings. Such properties might complement the cytotoxic potential of the apple pectins towards colon cancer cells since the enhancement of ROS production might lead to cellular death via triggering apoptosis. This observation is in apparent contrast with the numerous reports on the antioxidative activity of various pectin preparations (reviewed in ), includthocarpa . Pectinsthocarpa . It can Dioscorea opposite, which also attenuated the expression of IL-1β and IL-6 in colon tissues [The chronic inflammatory process is nowadays believed to lie at the root of the development of many progressive diseases, including cancer. Since the anti-inflammatory activity of pectins has been widely observed (for a review see ), it was tissues .Toll-like receptors (TLRs) play a key role in pro-inflammatory signaling networks due to their ability to recognize a variety of pathogen-associated products  and subsequent triggering of the production of inflammatory cytokines, co-stimulatory molecules, interferons, and chemokines . TLR4 isAnother essential regulatory protein with which pectins are known to interact is galectin-3. Gal-3 belongs to the lectin family and specifically recognizes β-galactosides  isolated from a Crohn’s disease patient was used in the experiments. Reduced adhesion of LF82 to cancerous epithelial cells in the presence of PX demonstrated the interference of pectin with E. coli adherence. LF82 adhesion is determined by mannose-sensitive type 1 fimbriae that bind to mannose receptors on the surface of intestinal epithelial cells. Pectins containing, among others, mannose groups, can interfere with the adhesion of LF82 to the intestinal epithelium that was also shown in our previous study on endo-cellulase-extracted pectin [Interaction of all dietary compounds with colonic cells might be modulated by the local microbiota. Therefore, it was checked whether enzymatically extracted apple pectins could affect the adherence of d pectin .In summary, we have clearly shown that the apple pectins PX and PCX exhibited antiproliferative, proapoptotic, and anti-inflammatory activities in colon cancer cells. Moreover, both pectins exhibited synergy with SN-38 in reducing the viability of cancer cells. As with the molecular mechanism of anticancer and anti-inflammatory properties of the pectins, the putative interaction with Gal-3 and TLR4 was suggested, respectively. Interestingly, PCX turned out to be a more active antiproliferative agent than PX, while their anti-inflammatory activity was comparable. Moreover, PCX also exhibited a stronger synergy with SN-38. This prompted us to attempt to analyze the features of three different enzymatically extracted apple pectins that might be important for their anticancer activity. PX was obtained by apple pomace treatment with endo-xylanase, PC with endo-cellulase, and PCX with both enzymes . The antSN-38 (7-ethyl-10-hydroxy-camptothecin), an active metabolite of irinotecan, was a product of Sigma-Aldrich . Stock solution of SN-38 was prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C. Stock solutions of pectins were prepared at a concentration of 10 mg/mL in double-distilled water. Stock solutions were diluted in a culture medium just before the experiments.Trichoderma viride. Isolation was performed with 50 U of enzyme per 1 g of pomace, at pH 5.0, in 40 °C, for 10 h, according to the method described by Wikiera et al. [Macromolecular apple pectin was extracted from dried apple pomace using endo-cellulase  and endo-xylanase  produced by filamentous fungus a et al. . Brieflya et al. . Molecul2, under standard conditions. The medium was changed twice a week. Confluent cultures were passaged using 0.25% trypsin. All experimental procedures were carried out in the log-phase of cell growth.The studies were performed on human colon cancer cell lines: HT-29, HCT 116, Caco-2 and FHC, obtained from ATCC collection. The cells were cultured in medium DMEM-F12 (PAA) supplemented with 1% antibiotics  and fetal bovine serum . The concentration of FBS in medium was 10% for HCT 116. HT-29, and FHC cells, and 20% for Caco-2 cells. Moreover, DMEM-F12 for Caco-2 cells contained non-essential amino acid solution (1×) . The medium for FHC cell line was additionally supplemented with 0.005 mg/mL transferrin, 100 ng/mL hydrocortisone, 20 ng/mL human recombinant EGF. The cells were cultivated at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed using MTT -2,5-diphenyltetrazolium bromide) method, as described previously [570 of treated cells/A570 of control cells) × 100%. The experiments were repeated three times.Cells were seeded on 96-well plates  24 h before the experiment. Then, the medium was removed, and a fresh medium containing studied pectins (PX or PCX) in the concentration of 0.05; 0.1; 0.2; or 0.5 mg/mL was added. In the experiments in which the combination of pectin and SN-38 was used, the concentration of PX or PCX was 0.2 mg/mL, whereas the concentration of the drug was 5 nM. The appropriate control containing DMSO in fresh medium was also prepared. The plates were incubated for 48 h at 37 °C in a humidified atmosphere with 5% COeviously . In gene491 of treated cells/A491 of control cells) × 100%. The experiments were repeated three times.The sulforhodamine B (SRB) assay  was modiThe combination index (CI) was calculated using CompuSyn software  according to the classic median-effect Equation (1), as was previously described by .(1)CI=D1 is the dose of drug 1 alone that inhibits a system by x%, (Dx)2 is the dose of drug 2 alone that inhibits a system by x%, and (D)1 + (D)2 are the doses of drug 1 and 2 in combination that also inhibit a system by x%. CI values below 1 represent synergism, CI value equal to 1 indicates additive effect , and CI values above 1 point to antagonism.In the Equation (1), (Dx)Combenefit is a software tool that enables the visualization, analysis, and quantification of substance combination effects . Data tag, 3 min, room temperature). The apoptosis was investigated using an Annexin-V apoptosis assay . To study cell cycle, the cells were fixed with hypotonic buffer containing propidium iodide (PI) at 100 µg/mL in PBS; 5 mg/L of citric acid; 1:9 Triton-X solution; RNase 100 µg/mL in PBS . The level of ROS was measured using a cell-permeable non-fluorescent probe, 2’,7’-dichlorofluorescin diacetate , which undergoes hydrolysis by cellular esterases into its polar form DCFH. Due to the intracellular ROS and other peroxides oxidation process takes place and DCFH turns into highly fluorescent compound 2’,7’-dichlorofluorescein (DCF). Cytometric analyses were performed using an Aria III flow cytometer  with FITC configuration  or with PE configuration , and at least 10,000 cells were counted. All experiments were performed in triplicate.In order to study cell cycle, an apoptosis and intracellular level of reactive oxygen species (ROS) flow cytometry method was implemented. The cells  were seeded onto 12-well plates 24 h before the experiment. Then, the medium was removed, and a fresh medium containing pectin (PX or PCX) at the concentration of 0.2 mg/mL with or without SN-38 at the concentration of 5 nM was added. The appropriate control containing DMSO in fresh medium was also prepared. The cells were incubated for the next 48 h at 37 °C. Further procedures was carried out as described previously . Brieflyg, 5 min, 25 °C). A further procedure was carried out as described previously [The activity of caspase-3 was analyzed using a commercially available kit , according to the manufacturer’s protocol. Cells were seeded  onto a 6-well plate in 2 mL of medium and incubated for 24 h at 37 °C. Then, the medium was removed, and a fresh medium containing pectin (PX or PCX) at concentration of 0.2 mg/mL with or without SN-38 at 5 nM was added. Control wells contained the cancer cells in fresh medium with DMSO. The cells were incubated for the next 48 h (at 37 °C), then scraped and centrifuged  to remove insoluble material. In order to generate MDA-TBA adducts, supernatant was mixed with tiobarbituric acid (TBA) and incubated for 60 min at 95 °C. Then, the samples were cooled to room temperature and added into a 96-well microplate for analysis. A microplate reader was used to measure the absorbance at 532 nm. In order to determine MDA concentration in the samples, a standard curve was used.The level of malondialdehyde (MDA), a product of lipid peroxidation which is a biological marker of oxidative stress, was analyzed using Lipid Peroxidation (MDA) Assay Kit , according to the manufacturer’s instructions with minor modifications. Cells were seeded  onto a 6-well plate in 2 mL of medium and incubated for 24 h at 37 °C. Then, the medium was removed, and a fresh medium containing pectin (PX or PCX) at concentration of 0.2 mg/mL with or without SN-38 at 5 nM was added. The cells were incubated for next 48 h (37 °C). After incubation, the cells were harvested and washed with PBS. The pellet of cells was suspended in Lysis Solution containing MDA Lysis Buffer and butylated hydroxytoluene (BHT). The cells were homogenized on ice and then centrifuged  in the concentration of 5 μM was added to each well 24 h before the experiment. This step was omitted in case of Gal-3 assay. Next, the cells were treated by the studied pectins at the concentration of 0.2 mg/mL for 48 h. IL-6, COX-2, TLR4, and Gal-3 were detected in cancer cells culture lysates using IL-6 Human ELISA Kit , Human COX-2 ELISA Kit , Human TLR-4 ELISA Kit , and Human Galectin-3 ELISA Kit , respectively. The studies were performed according to the manufacturer’s instructions. All experiments on the detection and quantification of selected proteins were repeated three times.The enzyme-linked immunosorbent assay (ELISA) method was used to detect and quantify cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), toll-like receptor 4 (TLR4), and Gal-3. First, the cells  were seeded in 96-well plates . In order to stimulate immune response lipopolysaccharide  isolated from a patient with Crohn’s disease was kindly provided by Dr. Arlette Darfeuille-Michaud . HCT 116 cells were infected with E. coli and incubated with PX or PCX in concentrations of 0.125, 0.25, and 0.5 mg/mL. Cells untreated with pectins and infected with bacteria constituted a positive control. After 2 h of incubation , the cells were washed with PBS and lysed with 0.1% Triton X-100. Serial dilutions of bacterial lysates were plated onto nutrient agar and incubated overnight at 37 °C to count bacterial colonies (CFU). The experiment was repeated three times in duplicate. The results are presented as the percentage of E. coli adhering to untreated cells, with negative control established to be 100%.The adherence of bacteria cells to colon cancer cells in the presence of pectins, PX or PCX, was investigated as previously described . An adheAdhesion images were taken under a light microscope after Wright–Giemsa staining. HCT 116 cells were cultured on slides in the wells of a 24-well plate. After 2 h of adherence experimentation, HCT 116 cells were washed and fixed with formaldehyde in the concentration of 4% . Then, formaldehyde was washed off and the cells were stained with Wright–Giemsa for 30 min. The dye was rinsed off, and the slides were dried and inspected under a microscope.t-test  with the use of Statistica 10 software.The values of measured parameters were presented as the means ± standard deviation (SD) from three independent experiments. The statistical significance was determined by Student Summing up, the antiproliferative, proapoptotic, and anti-inflammatory properties of enzymatically extracted apple pectins were demonstrated in colon cancer cells. Additionally, the synergy between pectin and SN-38 was discovered. The interaction of the apple pectins with Gal-3 and TLR4 was the molecular mechanism for their anticancer and anti-inflammatory effect. It was concluded that the relatively low molecular mass of PCX together with the relatively high proportion of RG I regions gave this pectin superior anticancer activity.


C. elegans in a microfluidic device for 3 days and analyzed temporal patterns of C. elegans activity by fractal analyses. The residence-time distribution of C. elegans behaviors shared a common feature with those of human and mice. Specifically, the residence-time power-law distribution of the active state changed to an exponential-like decline at a longer time scale, whereas the inactive state followed a power-law distribution. An exponential-like decline appeared with nutrient supply in wild-type animals, whereas this decline disappeared in insulin-signaling-defective daf-2 and daf-16 mutants. The absolute value of the power-law exponent of the inactive state distribution increased with nutrient supply in wild-type animals, whereas the value decreased in daf-2 and daf-16 mutants. We conclude that insulin signaling differentially affects mechanisms that determine the residence time in active and inactive states in C. elegans behavior. In humans, diabetes mellitus, which is caused by defects in insulin signaling, is associated with mood disorders that affect daily behavioral activities. We hypothesize that comorbid behavioral defects in patients with diabetes may be attributed to altered fractal scaling of human behavior.Fractal scaling in animal behavioral activity, where similar temporal patterns appear repeatedly over a series of magnifications among time scales, governs the complex behavior of various animal species and, in humans, can be altered by neurodegenerative diseases and aging. However, the mechanism underlying fractal scaling remains unknown. Here, we cultured  Such episodic bouts of behavior have also been observed in other vertebrates (mice4) and invertebrates . Activity time series of episodic behavioral bouts have non-periodic and intermittent patterns that appear repeatedly across a broad range of time scales. Self-similar geometrical patterns across time scales are called fractal patterns; therefore, activity time series of animal behavior are characterized by fractal geometry. Neurodegenerative disorders  or aging have been shown to alter the fractal scaling of various human behaviors11. These findings suggest that fractal scaling of animal behavior is regulated by neurophysiological mechanisms, which may be conserved among various animal species.In humans, ordinary daily activities12, and a reward evaluation for each object or event13 are thought to play important roles. Insulin signaling has been shown to affect mood and the reward system in mouse and human brains14. Mice with a brain neuron-specific knockout of the insulin receptor gene (NIRKO mice) did not show defects in neuron proliferation or death during brain development; however, they did show age-related anxiety and depressive-like behaviors15. In humans, nasal administration of insulin, which is mainly incorporated and functions in the brain without affecting blood glucose concentration in the periphery16, improved mood in both healthy individuals and patients with diabetes17. These results suggest that insulin signaling functions in mood control in the mouse and human brain. Insulin signaling, which has evolved in relation to the mood and reward systems in brain in higher animals, modulates the relation between olfactory stimuli and behavior in nematodes and flies14. Thus, insulin signaling is an evolutionarily conserved signaling system that coordinates external stimulation and animal behavior via neurosensory modulation. However, how insulin signaling affects the fractal scaling of animal behavior remains uninvestigated.Daily and social activities are affected by a broad range of neurophysiological states in the human brain. Among them, mood, an unconscious disposition to respond emotionally to objects or events encountered in lifeC. elegans behavior. Alternating switching between an actively moving state (“active state”) and an inactive state in episodic behavior is a common feature of various animal species. Therefore, we dissected the fractal scaling of C. elegans behavior on a two-state transition model5. Generally, kinetics that governs the state transition can be inferred from statistical properties, such as the frequency distribution and temporal correlation of experimentally measured residence times in each state. Inferred kinetics provides insights into the underlying mechanisms that drive the state transition. Through longitudinal videorecording of C. elegans swimming behavior, we found that state transitions between active and inactive states in C. elegans episodic behavior are driven by kinetics that determines residence times by following frequency distributions and temporal correlations with fractal properties. Therefore, we refer to the kinetics as “fractal kinetics”5.In the present study, we applied a genetic analysis of the fractal scaling of animal behavior by studying C. elegans with food bacteria. Specifically, we examined wild-type C. elegans cultured with or without nutrient source , as well as daf-2 and daf-16 nematode mutant strains cultured with nutrient source. The mutant strains have mutations in the insulin-like receptor gene (daf-2) and in the downstream forkhead transcription factor gene (daf-16)18. Our observations revealed that the fractal kinetics of C. elegans behavior is regulated by insulin signaling. Based on recent neuronal network modelling and molecular biological studies, we discuss the possibility that insulin signaling regulates neural activity in the brain to modulate fractal scaling of C. elegans behavior. We also discuss the applicability of this mechanism for mood disorders that are comorbid with diabetes mellitus in humans. We propose that fractal behavioral analysis can provide a more integrated clinical view of psychiatric symptoms in patients with diabetes, which may contribute to the development of new diagnostic indices and improvement of clinical treatment.In this study, we extended the function of the microfluidic device for culturing C. elegans behavior, we constructed a new microfluidic device composed of an array of 50 chambers for culturing individual animals by perfusing M9 buffer containing food bacteria  for 3 days5. By analyzing recorded movies using an image-processing program5, we obtained time series of behavioral activity with 105 time points  and the residence time in the inactive state  were measured alternatingly along an activity time series. In step 1, we obtained the residence time for the first round of the active state and the residence time for the first round of the inactive state. Similarly, in step 2, we obtained the residence time for the second round of the active state and the residence time for the second round of the inactive state. This measurement process continued such that in step vs.) round series” (DRS)  time series”. DRS in fed wild-type animals revealed that residence times in the active state varied from sub-seconds to 10 s, whereas residence times in the inactive state varied from sub-seconds to 100 s  or cultured with 1 g/L glucose  20. In DMA and its two-variable extension, detrending moving-average cross-correlation analysis (DMCA), when the fluctuation functions  at shorter round scale (72) Fig. a, and th68) Fig. a indicatRSs Fig. .Figure 3daf-2 and fed daf-16 mutants, respectively, Fig. To study the relation between active and inactive DRSs, we estimated the cross-correlation coefficients between two DRSs at various temporal scales . Fed wi,2s, Eq. . The neglong-range cross-correlation between active and inactive DRSs, we employed DMCA. In fed wild-type animals, Hurst exponents of a cross-correlated component between active and inactive DRSs at a shorter round scale  Fig. c indicat24. Therefore, we consider that a DRS with a strong fractal memory generated by an upstream fractal kinetics is provided to both the active and inactive DRSs as a pseudo-cross-correlated component via a vertical interaction mechanism. On the other hand, at longer round scale, daf-2 and fed daf-16 mutants, respectively; Fig. C. elegans behavior is discussed in the Supplementary Discussion  models26, including: stochastic NN models based on second-order phase transition 31, a deterministic model based on a feed forward-type NN model32, and another deterministic model at the “edge of chaos”33, indicating that power law neural avalanche dynamics is due to the collective dynamics of neuron in the brain. Together, these studies suggest that animal fractal behavioral scaling is derived from collective dynamics of neurons in the brain. In C. elegans, we found that the residence-time power-law distributions in the active and inactive states had the same distribution shapes and − 2 power law exponents  vs. dauer-formation defective (Daf-d) phenotypes for the daf-2 and daf-16 mutants, respectively35. Unexpectedly, we found that most of the Fractal Kinetics parameters summarized in Fig. daf-2 and daf-16 mutants , the average neuronal avalanche duration was elongated, such that the exponential-like decline disappeared from the frequency distribution observed in a brain slice cultured without the antagonist. Interestingly, with an inhibitory neuron antagonist, the frequency distribution at the longer time scale was beyond the power-law distribution, similar to what we observed in fed daf-2 and fed daf-16 animals  are involved in behavioral threat-reward decision making, although the involvement of insulin signaling in this process has not been studied in C. elegans42. The mechanism to determine the power law and exponential-like distribution of residence time in the active state shared in Fractal Kinetics A1 and A2 may be generated from the specific neuronal network containing D-MNs in C. elegans, as an analogy to the GABA inhibitory ganglia in amygdala in mammalians. Based on our findings, together with previous theoretical and experimental studies, we propose the hypothesis that insulin signaling activates GABA inhibitory neurons and alters the power-law neuronal avalanche distribution to be exponential-like, thereby changing the accompanying power-law residence-time distribution to be exponential-like in C. elegans behavior. With regard to Fractal Kinetics I and C, how they are generated and how their interactions in the kinetic regulatory pathway  and temperature-sensitive mutants of genes in insulin signaling, CB1370 daf-2 (e1370) and CF1038 daf-16 (mu86) were maintained on Nematode Growth Medium (NGM) agar plate at 15 °C.5, were fabricated by combining conventional photolithography and soft lithography methods44. For WormFloII, a polydimethylsiloxane (PDMS) chip and a bottom PDMS plate with 1-mm thickness were assembled using the oxygen plasma bonding method. Food bacteria suspension ) were supplied to the WormFloII, whereas M9 buffer with/without 1 g/L glucose were supplied to the WormFloI to maintain animals for observation at 0.4 ml/h with the Micro Ceram pump . Due to the structure of the junctional micro-slit channels in each chamber, an introduced adult animal in the chamber was held and its offspring flowed off just after hatching from the chamber. We confirmed the absence of offspring at the end of recording.Two microfluidic devices, WormFloII and WormFloIdaf-2 and daf-16 mutant strains, 24 °C, for one day and transferred to the microfluidic device that was soaked in M9 buffer in a 15-cm-diameter glass dish at room temperature. Then, the 15-cm-diameter glass dish containing the microfluidic device was installed in a temperature-controlled aluminum box maintained at 25 °C5. Temperature acclimation at the start of recording and later maintenance for 3 days were confirmed by direct temperature monitoring5. Animals were observed under blue-light-cut illumination by a macroscope with an apochromat objective lens (1 ×)  and recorded in an H264 compressed movie5. Animal swimming activity was measured by counting pixels in a bitmap image, in which animal movement was determined by comparing the image at the previous time frame  were picked up and cultured at non-permissive temperature for the For DMA, the fluctuation function For DMCA, the cross-fluctuation function Due to rejection of the normality hypothesis for scaling exponents in DMA and DMCA, and the power-law exponents of the residence-time distribution, the non-parametric Wilcoxon rank sum test was employed for pairwise comparisons between groups. In pairwise comparisons, the Benjamini and Hochberg method for correcting the false discovery rate (FDR) was used to deal with multiple testing problems.46.A previous version of this manuscript was published as a preprintSupplementary Legends.Extended Data Fig. 1.Extended Data Fig. 2.Extended Data Fig. 3.Extended Data Fig. 4.Extended Data Fig. 5.Extended Data Fig. 6.Extended Data Fig. 7.Extended Data Fig. 8.Extended Data Fig. 9.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.Supplementary Discussion.


Ancylostoma duodenale  infection was measured after starting treatments with piperazine. This study aims to determine the impact of cytokine production after infection with A. duodenale before and after treatment with piperazine. Blood and stool samples of 50 patients with A. duodenale infection and 28 healthy individuals (control) were collected. In this study, IFNγ, IL-5, IL-12, and IL-13 in serum (using ELISA-based methods) were measured. Stool samples were examined using the Kato-Katz technique to detect A. duodenale parasites. Blood and stool samples were analyzed 14 days after starting piperazine treatment for A. duodenale infection. The medium concentration of IFNγ, IL-5, IL-12, and IL-13 in the serum samples with A. duodenale infection is higher than that of the control group. IFNγ, IL-5, IL-12, and IL-13 levels were significantly higher in the infected individuals  than the control group . Also, piperazine treatment can significantly reduce cytokines levels . This study shows that piperazine treatment can reduce cytokines profiles in patients with A. duodenale infection.Cytokine response to  Ancylostoma duodenale  infection caused by intestinal helminthiasis is caused by hookworms. Most cases are concentrated in tropical and subtropical countries  [A. duodenale infection is related to the production of pro-inflammatory cytokines IFN-γ, Th1 cells [A. duodenale infection related to Th2 immunity response can be distinguished through the production of IL-5 and IL-13, enhancing IgE and IgG4 antibody response [A. duodenale infection are the complex connections of cytokines and response mediators, but these complex connections are fairly known (regardless of the frequency of these infections and numerous treatments) [rldwide) . Iron deh1 cells . In the h1 cells . A. duodPiperazines are a broad class of chemical compounds with several uses in industry and medicine and are pharmacologically identical to the normally occurring neurohormone inhibitors. It is available as a well-known medicine used as a treatment option against anthelmintic infections, and it is very useful to control intestinal parasites . In diffA. duodenale were analyzed and compared to the immune response of the control group. Furthermore, we compared cytokine serum levels pre and post piperazine treatment.In this study, the levels of IFNγ, IL-5, IL-12, and IL-13 cytokines in the blood serum of patients infected with Ancylostoma duodenale infection . The intensities of A. duodenale infection were determined through Kato-Katz fecal thick smear technique [A case-control study was conducted with patients, in Al-Furat Al-Awsat teaching hospital, in Al-Najaf province, from January 2021 to May 2021. Patients were 30 males and 20 females, aged 18-45, diagnosed with echnique , and theAfter obtaining consent, a 5 ml blood sample was withdrawn from each participant via vein puncture. The plasma was separated by centrifugation at 1000 rpm for 15 min using 5% EDTA anticoagulation tubes. The separated plasma samples were then stored at -70°C for the determination of cytokine levels.The cytokines level yields for all supernatants were detected . Cytokines examination was done using commercially ready ELISA kits, which have dual cytokines specific monoclonal antibodies, following the manufacturers' recommendations and instructions .Data were analyzed, and the mean±standard deviations were calculated. T-test was used to compare the means of cytokine concentrations for patients and control groups. The data analysis was performed using SPSS software, where the rating equation models for data, distinguishing standard errors, to assess any significant differences in the cytokines levels pre and post piperazine treatment, using Rubin's rule .A. duodenale were higher than in the control group. There were significant differences in the concentrations of IFNγ (P<0.001) and IL-13 (P<0.05) between the 2 groups , IL-5  and IL-12 , after treatment with piperazine and worms were expelled. There was no significant IL-13 level alteration post piperazine treatment .A. duodenale infection. In addition, the eggs of this parasite can stimulate a rapid increase in IFNγ levels corresponding to the immune response. The level of IL-5 stimulation occurs 5 days after infection when the parasite is thought to be in the luminal phase [A. duodenale, can lead to the continuation of infection and rise of IL-12 level. IFNγ is a representative of the Th1 cytokine implicated in the rescue of the interaction of cellular pathogens, where the cytokines act on the regulation of antibody output, and MHC increases type II and energizes macrophage output oxygen-free radical and IFN-γ [This study shows a significant increase in IFN-γ, IL-5, IL-12, and IL-13 in serum levels after nd IFN-γ .A. duodenale. These reactions could signal the powerful immunomodulatory effects of A. duodenale in patients, thus directing the immune system to an extra immune response appropriate for cytokines' existence [The anti-inflammatory mediator, such as IL-12, is substantial in saving IFNγ levels to lower the host's potential reverse effect of a long time high IFNγ level . These rxistence . Eotaxinxistence .This study showed a significant reduction in IFN-γ, IL-5, and IL-12 levels in the plasma of patients after treatment with piperazine. Worm expulsion relieves the immune compression and is responsible for increasing cytokine levels, which is caused by returning the cytokines to the rest state. This was not discussed in other studies, like studies that boost the immunity of Th2 cytokine response followed by piperazine treatment in patients with schistosomiasis .A. duodenale group, pre, and post-treatment. The production of enzymes that inactivate exotoxins may be the strategy used by helminths to prevent the induction and effect of eosinophil in the site of infection .Participants received written informed consent before participating in the study.SAS contributed to data collection. HAS contributed to writing the original draft, methodology, and editing. SAS contributed to conceptualizing the study. ZFA contributed to data analysis and data curation.


Recently, great attention has been paid to developing new drug delivery systems to manage the rate, time, and site of drug release. We aimed to design a novel drug delivery system to support targeted and gradual delivery of levothyroxine sodium.In vitro drug release into the PBS was measured at different concentrations of the triblocks for one month. The triblock copolymers of PLA-PEG-PLA and PLGA-PEG-PLGA were constructed using the ring-opening copolymerization method and then purified and characterized by 1H-NMR, DSC, and GPC techniques. The phase transition temperature of the polymers was determined, and levothyroxine sodium stability was investigated in a phosphate-based buffer (pH 7.4). g points of -8.19 °C and -5.19 °C for PLA-PEG-PLA and PLGA-PEG-PLGA, respectively. Stability tests showed that during one month, most of the triblocks’ masses degraded at 37 °C while levothyroxine sodium remained stable. Initial burst release of the drug in both copolymers is inversely correlated with the concentration of the polymer. Evaluation of drug release for 35 days showed that PLA-PEG-PLA had a slower drug release rate than PLGA-PEG-PLGA.The results of NMR and GPC showed successful fabrication of the copolymers with low molecular weight dispersion and TConsidering the low initial burst release, as well as continuous and long-term release kinetics of PLA-PEG-PLA and PLGA-PEG-PLGA copolymers, they can be used to gradually deliver levothyroxine sodium, obviating the need for frequent administrations and concerns over drug-food interactions.  In recent decades, biodegradable hydrogels have acquired great attention because of their ability to protect the structure of unstable molecules such as proteins. These hydrogels have high water content and are hydrophobic intelligent materials that are responsive to various stimuli. The three-dimensional structure of hydrogels confers on them suitable mechanical stability. Furthermore, they are implantable because of their acceptable safety and biocompatibility. Hydrogels, due to considerable water content and high porosity, can enclose and then release large hydrophilic compounds such as peptide-based drugs. Because of these features, hydrogels are used by scientists for designing slow-release drug delivery systems.Intelligent hydrogels  respond to natural stimuli by modifying their structural and physical properties, and permeability. These stimuli can be divided into three main categories: physical , chemical , and biological  -5.Temperature-responsive hydrogels are known for their ability to swell and shrink depending on temperature variations . In factin situ gel-forming hydrogels, which are actually aqueous mixtures of gel precursors and low-viscose biologically active materials, undergo gelation in response to changes in factors such as temperature, pH, and the type of the solvent when they are injected into the body. One of the most important advantages of these hydrogels is that they do not require surgery to be delivered to the body (in contrast with implantable hydrogels). Besides, stimulus-responsive hydrogels can be loaded with different types of drugs  of the drug is reached in about two hours after administration, while in those with hypothyroidism, this time is close to three hours, which can even be extended by consumption of some foods  have been developed as slow-release drug delivery systems. These systems also contain N-methyl pyrrolidone (NMP), which is frequently used as a biocompatible water-miscible organic solvent. In aqueous milieu, NMP and water start to diffuse into the release medium and the polymer matrix, respectively . The contact of water with the matrix causes the polymer to precipitate, resulting in solid polymeric implant formation (® (PLGA-based leuprorelin acetate), Doxirobe® (PLA-based doxycycline hyclate), and Nuflor® (PEG-based Florfenicol)  . A disadfenicol) . The usefenicol) .et al. who investigated the drug release in vitro for three weeks . Due to presence of glycolide in its structure, PLGA-PEG-PLGA is more hydrophilic than PLA-PEG-PLA, which is thought to affect the release of levothyroxine sodium. Actually, the hydrophilicity effects of PLGA and PLA blocks on the rate of the release process of sodium levothyroxine from the NMP containing gel system have been investigated. In addition, drug release patterns, drug stability, and other features of PLA-PEG-PLA and PLGA-PEG-PLGA triblock co-polymer systems were compared , N-Methyl-2-pyrrolidone, and 3-  - 2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich. Levothyroxine sodium was purchased from Iran Hormone Co. (Iran). Cell culture medium (RPMI1640), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin were purchased from Gibco (Germany). Vivitrol® was obtained from Alkermes (USA), and other materials were from Merck (Germany).MethodsSynthesis of triblock copolymersPolymer fabrication in microwave: PLA-PEG-PLA and PLGA-PEG-PLGA were synthesized using the ring-opening polymerization (ROP) method. Initially, to dehydrate PEG, the polymer was placed into a microwave for 10 min (100 watts). Then lactide (for synthesis of PLA-PEG-PLA) or the combination of glycolide: lactide  was added to a balloon containing PEG (the mass (g) ratio of 10: 3 for the middle polymer (PEG) and the side polymers, respectively). The mixture was melted at 130 °C. Finally, 10 μl of pure Sn (Oct)2 catalyst was added to the reaction, and incubation continued for 10 min at 130 °C. The general scheme of the synthesis process of the triblock polymers (PLGA-PEG-PLGA and PLA-PEG-PLA) has been shown in Triblock copolymer purification°C, and then the copolymer container was placed on ice (4 °C) to generate the sol phase. In this condition, unreacted materials and the copolymer would be dissolved. Next, the temperature was elevated up to 65 °C to allow the fabricated polymers to precipitate, while unreacted materials remained dissolved in the supernatant. The supernatant was then discarded. These steps  were repeated three times to purify the polymer. After purification, lyophilization was performed to completely remove the water. The polymers fabricated were initially placed in a freezer at -20 °C for 24 hr and then transferred into a freeze-dryer (Purification was performed by dissolving the copolymers (PLA-PEG-PLA or PLGA-PEG-PLGA) in cold water and then precipitating impurities by immersing them in hot water. Twenty-four hours after fabrication, the synthesized copolymers were immersed into deionized water at 37 ze-dryer . Fabrication efficiency calculation For this purpose, the weight of the dried product was divided by the total mass of the materials used at the onset of the reaction. Triblock polymer identification and characterization 1H-NMR To identify the copolymers, the presence of lactide, PEG, and glycolide subunits (for the PLGA-PEG-PLGA triblock copolymer) was investigated via 1H-NMR spectroscopy. First, the samples were dissolved in deuterated chloroform, and then their spectra were determined at 300 MHz. To determine their characteristics, the obtained spectra were compared with a reference . Molecular weight determination by gel permeation chromatography The distribution of molecular weight (Mw) and molecular mass of the fabricated triblock polymers were determined by Gel Permeation Chromatography (GPC) using the tetrahydrofuran solvent at a flow rate of 1 ml per minute. Polystyrene was used to calibrate the system before sample injection, and a solvent was used to wash out the sample injected inside the separating column.Determination of phase change temperature°C to 55 °C (0.5 °C per minute). After each step, to reach a balanced temperature, a 2-minute lag phase was considered. Following each temperature rise/stop phase, the sample-containing tubes were completely inverted to inspect gel formation (evidenced by the sample remaining attached to the bottom of the inverted tube). The gelation temperature was recorded for each sample. Then the temperature was increased in the same manner until the polymer precipitated in the tube, forming a two-phase system. The temperature at which the sample turned into a two-phase compound was also recorded as the polymer’s precipitation point. The experiment was repeated three times for each sample.To determine the phase change temperature , the tube inversion method was used. First, 10-50% concentrations of the copolymer’s aqueous solution were prepared in a PBS buffer (pH 7.4) and incubated in the refrigerator for 24 hr, while stirring gently via a magnet until the polymer was completely dissolved. Then a certain volume of each sample was transferred to similar tubes that were placed in a water bath with the temperature gradually increasing from 25 DSC analysis°C to +60 °C (10 °C rise per minute) and were recorded under nitrogen gas flow versus empty aluminum crucibles as the reference.DSC analysis was performed for both PLGA-PEG-PLGA and PLA-PEG-PLA triblock polymers. For this purpose, 3 mg of the samples were placed into special aluminum crucibles, and the thermal behaviors of the samples were within the range of -60 Levothyroxine sodium standard curve A 10 mg/ml levothyroxine sodium stock solution was prepared by accurately diluting to 1000 ml with PBS, and other standard samples were prepared by making ½ dilutions. Afterward, 50 μl of each sample (in a concentration range of 0.0048 to 10 μg/ml) was injected into the HPLC (in triplicate). Finally, retention time and the area under the peak were recorded. Then the graph of concentration vs the area under the peak was plotted.Drug quantification by HPLC °C and 255 nm, respectively. The injection volume was 50 µl, and the infusion was repeated three times. Different concentrations were prepared and the peak absorbance of levothyroxine sodium was determined. At specified times, levothyroxine sodium was measured to determine the peak value. According to the area under the curve and the equation obtained from the graph, the drug’s concentration was determined in each sample.In this experiment, the mobile phase was the 40%:60% acetonitrile: water solution, to which 0.5 ml of phosphoric acid was added. The Buchner funnel and a vacuum pump were used to separate tiny particles. The flow rate of the mobile phase was 1 ml per minute, and temperature and wavelength were set at 25 Levothyroxine loading in copolymersFor loading the drug into the polymeric structure, levothyroxine was simply dissolved in the polymer solution. For this purpose, solutions of copolymers were prepared at the 30, 40, and 50% w/w concentrations in the NMP solvent, and then 6 mg levothyroxine sodium was added to each solution and completely dissolved by sonication for 2 hr.Viscosity Assessment °C. Finally, the supernatant was removed, and the viscosity of the precipitate was measured.To evaluate the viscosity of the triblock copolymers, 30, 40, and 50 % w/w concentrations of the copolymers were prepared in the NMP solvent. To determine the viscosity of each sample, 3 ml PBS buffer was added to 1 ml of the NMP-polymer mixture, and the samples were incubated for four days at 37 Drug release examination °C. Then 10 ml of PBS  was gradually added to the test tube. For investigating gelation, the height of the tube’s constituents was marked; the tips of the tubes were tightly closed, and they were transferred to a shaker water bath  to prevent the formation of a diffusion layer. Subsequently, at specific time intervals , 1 ml of the surface buffer, which contained the drug released, was withdrawn. The volume removed was replaced with 1 ml fresh buffer. Sampling was conducted at longer intervals during the last days because of a decline in the release rate. Finally, levothyroxine sodium level in each sample was determined via HPLC, and a standard graph (cumulative drug release vs time) was prepared.In order to evaluate the drug release rate, 1 ml of each of optimally prepared drug-containing formulations was transferred into a test tube and then placed in a water bath for five minutes to reach 37 n was calculated according to the following formula (The percentage of the drug released until t formula :n = Cn Vt + ∑ C (n-1) VsMn = True (cumulative) amount of nth sample (μg/ml)Mn = Apparent concentration (read) of nth sample (μg/ml)Cs = Volume removed from the sample (ml)Vt = Total volume of the release medium (ml)V(n-1) = Total apparent concentrations (read) of samples between 1st and n-1 (μg/ml)∑C Degradation features of triblock copolymers°C. Each sample was withdrawn at a specified time; the supernatant was gently removed, and the precipitate was kept at room temperature. On day 31st and after the last sample was processed, all the samples were transferred into a freeze-dryer to allow for complete drying before weighing the microtubes. The weight reduction rate reflected polymer degradation, according to which a degradation vs timetable was prepared.To perform the degradation test, weight changes of the synthesized triblock polymers were determined 37 °C after 1, 2, 3, 4, 5, 7, 10, 13, 16, 19, 22, 25, 28, and 31 days. A separate microtube was designated each time for weight measurement. A specific amount of the polymer was added to the microtube, and then the weight of the microtube was measured. The change of the wight of the microtube containing the polymer at the end of the degradation test corresponded to the change of the mass of the polymer. After weighing the microtube at the end of the test, one milliliter of deionized distilled water was added to each sample, and all the microtubes were transferred into a water bath at 37 The effects of the triblock copolymers on the viability of animal cells in vitro2 and 95% humidity at 37 °C until reaching the growth phase. To detach the cells from the bottom of flasks, 0.25% trypsin was used. After two passages, 10,000 cells (at the growth phase) were seeded in the wells of a 96-well plate and incubated overnight. Four wells were assigned to each copolymer formulation. The copolymer was mixed with an equal volume presented to the cells for either 24, 48, or 72 hr. After this step, 10 µl of the MTT solution (5 mg/ml in PBS) was added to each well, and the mixture was incubated for 4 hr at 37 °C. The culture medium was then removed to assess emerged blue-colored sediments. DMSO (100 µl) was added to each well, and the plate was placed on a shaker for five minutes. Finally, the absorption of the resultant solution was read at 570 nm (versus 630 nm as the background). Finally, the viability of the cells was estimated based on the absorption percentages of copolymer-treated cells vs untreated control cells. The experiment was repeated at least three times for each sample, and the results were presented as mean±SD. Relative cell death (R) was calculated using the following formula  and 1% penicillin (100 IU/ml)-streptomycin (100 mg/ml). Cell cultures were placed into an incubator with 5% CO formula :test / Acontrol) × 100] R = [. Finally, the IC50 values of the formulations were calculated using the GraphPad Prism software package.In this formula, AStatistical analysisStatistical tests  were conducted in GraphPad Prism software package.Ethical considerations This study was approved by the institutional ethical committee  under the code of IR.MUMS.PHARMACY.REC.1397.001.The synthesis yield of PLGA-PEG-PLGA and PLA-PEG-PLAThe PLGA-PEG-PLGA (with a lactide to glycolide ratio of 7:1) and PLA-PEG-PLA triblock copolymers were synthesized via the ROP method using a three-neck balloon. The synthesized triblock copolymers appeared light yellow and had semi-solid stickiness and viscosity. The synthesis yields of PLGA-PEG-PLGA and PLA-PEG-PLA were 83.4% ±2.65 and 85.1% ±2.41, respectively. Identification and characterization of triblock copolymers1H-NMR studies1H-NMR was used to determine polymer structure and ratios of lactide (LA) to glycolide (GA), as well as PEG 1500 to PLGA 1500. The spectrum of PLGA 1500-PEG 1500-PLGA1500 has been depicted in 3 group of lactide and the -CH2 of glycolide, respectively. The 4.34 ppm peak represented the PLGA-bound CH2 of PEG. The peak around 3.65 ppm was related to another -CH2 group in PEG, which due to its proximity to the -CH3 peak, appeared as a three-horn. The peak around 1.58 ppm was also related to the -CH3 group of lactide. Finally, the 7.29 ppm peak was attributed to the solvent (deuterated dichloromethane). 1H-NMR spectrum of PLA-PEG-PLA has been shown in The Molecular weight determination by GPC 1H-NMR analyses have been represented in Phase transition temperatureThe sol-to-gel phase transition temperature and precipitation temperature for PLGA-PEG-PLGA and PLA-PEG-PLA triblock copolymers have been shown in DSC Analysis g of PLA-PEG-PLA was -8.19 °C, and Tg of PLGA-PEG-PLGA was -5.19 °C, and its Tm was 27.46 °C.The thermograms of the fabricated triblock copolymers have been demonstrated in Levothyroxine sodium stability °C for one month.Levothyroxine sodium stability, according to HPLC, has been indicated in The syringeability and viscosity of synthesized copolymersBoth of the triblock copolymers represented good syringeability and could easily pass through a 25G syringe. The viscosity values of PLGA-PEG-PLGA and PLA-PEG-PLA triblock copolymers were obtained at 0.075±0.005 and 0.070±0.004 (Pa.s), respectively, indicating acceptable viscosity for subcutaneous injection. Drug release studiesThe drug release curves of the PLGA-PEG-PLGA were shown in our previous work  , and PLAP<0.0001).2) were compared .Triblock copolymer effects on the viability of animal cells Due to its interactions with sodium, calcium, and other minerals, levothyroxine sodium should be used daily and with an empty stomach. In addition, either forgetting to take the medicine in a timely manner or being unaware of the correct way of consumption leads to receiving inappropriate doses of the drug. On the other hand, due to the variable absorption rate (40 to 80%) and relatively low bioavailability (65%) of orally consumed levothyroxine sodium, it is not possible to accurately predict the drug dose entering the blood. Moreover, daily consumption of the drug requires the patient’s full cooperation. To solve these problems, it is required to design a system that can gradually release the drug in a controlled manner within a long period of time even following only one administration .Traditional drug delivery systems, which are based on oral, dermal, and mucosal routes, although suitable for the drugs, are not satisfactory for delivering a wide range of other drugs, such as protein-based and hormonal drugs, especially when long-term administration is required. Other disadvantages of traditional approaches include the fluctuations of drug plasma concentration, drugs’ instability and low solubility, and non-targeted drug delivery. Novel drug delivery systems, on the other hand, allow for targeted long-term drug delivery and drug release, reducing the dosage and side effects of drugs and boosting patient compliance, especially those with chronic diseases .et al. used porous silicon films (as containers) to develop subcutaneously injectable controlled-release (during 60 days) formulations of levothyroxine sodium  that released levothyroxine sodium slowly and continuously upon oral administration over the time-examined period . The monomers are then joined together sequentially to form a novel polymer. To confirm the successful fabrication of the triblock copolymers, they were characterized using In response to temperature, dispersions of polymers in buffer generally take one of the following three phases: sol (completely liquid and fluid), gel , and precipitated . We observed that by increasing the concentration of the copolymer, the sol-gel transition temperature decreased while the precipitation temperature raised. Increasing the temperature and polymer concentration led to micelle formation and facilitated the gelation process. This behavior of some polymers is somehow attributable to their amphiphilic structure  . In the g values of -8.19 °C and -5.19 °C for PLA-PEG-PLA and PLGA-PEG-PLGA, respectively. The higher temperature of the latter can be due to the presence of glycolide in PLGA, leading to the formation of a more crystalline network compared with PLA. The thermograms of the two fabricated triblock copolymers showed TDegradation analysis during 31 days showed a higher weight loss  for PLGA-PEG-PLGA compared with PLA-PEG-PLA, which can be attributed to the presence of glycolic acid in the former and therefore its more degradability in exposure to humidity .Drug release from the triblock copolymers synthesized into an aqueous medium containing PBS buffer was investigated during the first 24 hr after injection  and afterward. In drug delivery systems with a high initial release rate, a large amount of the drug is released during the very first moments, which can cause toxicity. In this study, different ratios of PLGA or PLA to PEG were tried to guarantee the lowest initial burst release. In this study, the initial burst release was determined for six different formulations, among which the PLGA-PEG-PLGA formulation containing 30% hydrogel revealed the highest release rate (17.3±1.2%) within the first 24 hr post-injection. A negative correlation was seen between the concentration of the triblock and the initial burst release rate. Accordingly, the initial burst release at 40% of PLGA-PEG-PLGA was 9.57±2.03% that decreased to 6.52±1.3% at the 50% concentration of this copolymer. Compared with PLGA-PEG-PLGA, the initial burst release of PLA-PEG-PLA was lower, retrieving the values of 12.0±1.7%, 7.06±1.2%, and 4.48±0.7% for 30%, 40%, and 50% concentrations of the triblock, respectively. Overall, the PLGA-PEG-PLGA triblock copolymer delivered a higher initial drug release rate than PLA-PEG-PLA. This observation can be justified by the more hydrophobic nature of PLA compared with PLGA. The hydrophobic nature of PLGA respective to PLA causes this compound, along with its solvent (NMP), to slowly enter the aqueous phase upon being injected into a simulated body environment, blunting initial drug release during the first 24 hr. et al. who employed the two PNIPAM and PVA polymeric systems to deliver levothyroxine sodium via the skin, the initial drug release rate into PBS at 37 °C was 73% for PNIPAM and 48% for PVA during the first 15 min . In a study by Rostami  was 81% , in compectively . Moreoveectively .In the zero-order drug release kinetic model, the drug is supposed to travel a constant distance by passing through a membrane with a constant thickness to reach the target. In the matrix-based diffusion model, the drug that is closer to the outer surface is initially released, followed by the drug located into deeper segments of the system. In other words, the thickness of the diffusion channel is greater in the central parts of the system . For the thickness of the drug passage channel to remain constant, the polymer’s degradation should be equivalent to the drug release rate. So, it can be concluded that in matrix systems for zero-order release, drug release should be mainly based on polymer degradation while in the Higuchi model, release occurs mainly via diffusion through matrix. In this study, for both of the synthesized triblock copolymers, the regression coefficient was close to one for zero-order kinetic.Regarding the Korsmeyer-Peppas model, if the “n” index is ≤0.43, drug release is supposed to follow the penetration model (or Fickian diffusion); if 0.43 ≤ n <0.89, the pattern follows non-Fickian diffusion, and if n ≥ 0.89, drug release is based on polymer degradation . In this study, the “n” index for both of the triblock polymers was ≥0.89, confirming the zero-order release pattern and supporting the findings of polymer degradation analysis.The drug release kinetics of both of the triblock copolymers followed an almost constant and linear pattern except during the first few hours, offering controlled drug-delivery systems with relatively constant drug release rates corresponding to polymer degradation. Although we monitored drug release for 35 days, levothyroxine sodium was completely depleted from all six formulations, independent of the copolymer’s concentration, during 21 days.°C) and under 28 °C, respectively. The triblock copolymers began to transform into a gel, from the above-mentioned temperatures and remain in the gel phase to 60 °C and 63 °C in PLGA-PEG-PLGA and PLA-PEG-PLA triblock copolymers, respectively. It is shown that the two triblocks gel at body temperature (37 °C). The release of the levothyroxine sodium entrapped in these copolymers was slow and continuous. The polymers were biodegradable and biocompatible and had no toxicity against human living cells (L929 skin fibroblast cell line). They were also stable in an anhydrous environment for one month, showing no significant weight loss. After completely releasing the drugs loaded, these polymers are degraded in body fluids and do not need surgery for their removal. The release of levothyroxine sodium from these systems followed a relatively linear pattern with a low initial burst release within one month. The PLGA-PEG-PLGA triblock copolymer delivered a higher initial drug release than PLA-PEG-PLA. This observation can be justified by the more hydrophobic nature of PLA compared with PLGA.At the concentrations of 30%, 40%, and 50%, PLGA-PEG-PLGA and PLA-PEG-PLA triblock copolymers were in the sol phase at room temperature (25 JM and FH contributed with conceptualization, supervision, original draft preparation, reviewing, and editing. EK and SNRS helped with writing, reviewing, and editing. BK helped in writing the original draft preparation. HK supervised and helped with writing, reviewing, and editing. FO contributed with conceptualization, supervision, original draft preparation, reviewing, editing, and graphics and picture preparation. No conflicts of interest were reported by the authors.


Depression has a high prevalence among European countries. Several instruments have been designed to assess its symptoms in different populations. The Hopkins Symptom Checklist 25 (HSCL-25) scale has been identified as valid, reproducible, effective, and easy to use. There are short versions of this scale that could be useful in Primary Care (PC) settings, but their psychometric properties are unknown.To assess in PC patients the psychometric properties and diagnostic accuracy of the Spanish version of the HSCL-10 and the HSCL-5 consisting of 10 and 5 items, respectively.A multicenter, cross-sectional study was carried out at six PC centers in Spain. The HSCL-25 was administered to outpatients aged 45–75 who also participated in the structured Composite International Diagnostic Interview (CIDI). HSCL-10 and HSCL-5 were assessed and compared to HSCL-25 regarding total score correlation, internal consistency, and criterion validity against the gold-standard CIDI. This is a methodological study from a secondary data analysis and the primary data has been previously published.Out of 790 patients, 767 completed the HSCL-25 and 736 the CIDI interview (96.0%). Cronbach’s Alpha was 0.84 for HSCL-10 and 0.77 for HSCL-5. The known-group method and confirmatory factor analysis were acceptable for the establishment of construct validity. Sensitivity was 79.7%  for HSCL-10, and 78.0%  for HSCL-5, whereas specificity was 83%  for HSCL-10, and 72.8%  for HSCL-5. Area under the curve against CIDI was 0.88  for HSCL-10, and 0.85  for HSCL-5. Optimum cutoff point calculated with Youden Index was 1.90 for the HSCL-10 and 1.80 for the HSCL-5.HSCL-10 and HSCL-5 are reliable and valid tools to detect depression symptoms and can be used in PC settings. Mental health is an issue of increasing concern in Spain and other European countries and represents a considerable percentage of Primary Care (PC) consultations. The clinical diagnosis of mental disorders is based on the symptoms of the Diagnostic and Statistical Manual of Mental Disorders (DSM). In addition, there are many questionnaires available which assess mental health diseases in populations and are frequently employed in PC . Such inThe United States Preventive Services Task Force (USPSTF) recommends universal screening for depression in the general adult population  as it haUp to 75% of patients with depression are treated exclusively in PC . NeverthThe ideal questionnaire should be reliable, valid, ergonomic, and easy to use for both patients and healthcare professionals. It should be validated according to the Consensus-based Standards for the Selection of Health Measurement Instruments (COSMIN)  for the The Hopkins Symptom Checklist 25 (HSCL-25)  is a widThe HSCL-25 is a short version of the Symptom Checklist 90-Revised (SCL-90-R) . There aThe aim of this article is to report the psychometric properties and diagnostic accuracy of the Spanish versions of the HSCL-5 and HSCL-10 for their use as rapid and accessible depression screening instruments in PC.The present study was based on data from a cross-sectional multicenter study designed to validate the HSCL-25 in a Spanish PC population. This is a methodological study from a secondary data analysis and the primary data has been previously published . ParticiParticipants were recruited by consecutive sampling of patients attending the PHC for any reason during a 6-month-period in 2017. They were asked to complete sociodemographic data  and the self-administered HSCL-25 questionnaire (and other questionnaires/forms related to the EIRA study). Afterward, trained professionals, blinded to the HSCL-25 score, conducted the gold-standard CIDI interview with all participants.The HSCL-25 is a widely used, self-administered questionnaire designed to measure anxiety and depression symptoms , 11 and Items belonging to HSCL-25, HSCL-10, and HSCL-5 are shown in The CIDI is a well-known, standardized interview designed by the World Health Organization (WHO) based on the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) and the International Classification of Diseases-10 (ICD-10) criteria . It is aAs the diagnose of depression is a clinical interview performed by a trained professional and conducted using the DSM criteria, the CIDI is considered the gold-standard in the present study.The study was developed according to national and international legislation (the Declaration of Helsinki and latest versions). The protocol was evaluated by the IDIAP Jordi Gol Ethical Research Committee  and by the corresponding regional governments. Written consent was obtained from the participants, and the questionnaires were codified with an identification number to protect anonymity and confidentiality.Analysis was conducted with STATA version 15. Missing values for the HSCL-25 were replaced with the individual mean for the rest of the items. Subjects with ≥50% missing items were excluded.Total score was calculated for HSCL-25, HSCL-10, and HSCL-5 for the total population and in relation to gender and age categories, and by the following sociodemographic groups: marital status, education level, and current employment.Reliability of the HSCL-10 and HSCL-5 was analyzed by calculating the Cronbach’s Alpha Coefficient and for each of the two depression and anxiety subscales. A value of ≥0.7 was considered adequate . Cronbact-test was performed, a significant result (p < 0.05) was considered satisfactory . Participants’ mean age was 58.4 years (± 8.2), 54.4% were women, and there were no significant gender differences among age categories. There were statistically significant differences in total scores for the three versions regarding gender. Women scored higher with a minimum difference >0.25 points. There were also statistically significant differences in the total scores of the HSCL-25 and HSCL-10, but not the HSCL-5, with respect to age and marital status. No differences were observed regarding education level or current occupation.Cronbach’s Alpha was 0.8417 and 0.7712 for the HSCL-10 and HSCL-5, respectively. When analyzing the two dimensions separately, this value was higher for the depression dimension than the anxiety one. These values, and the value of the coefficient without the item, are depicted in The most consistent item for the HSCL-10 was 17 “Feeling blue” followed by 22 “Feeling that everything is an effort.” The least consistent was 25 “Sleep disturbance” followed by 1 “Being scared for no reason.” Item 25 “Sleep disturbance” had a Cronbach’s Alpha without the item of 0.8424, as a result, this item worsened the reliability of this version, as this value was above 0.8417. In the HSCL-5, item 17 “Feeling blue” was also the most consistent whilst two “Feeling fearful” was the least consistent although without affecting reliability.t = 7.76; p < 0.001). Results were in the same direction with the HSCL-5, total score was significantly higher in women  than in men .The known-group method analysis showed that the total score of the HSCL-10 indicated that women had significantly higher scores  than men , and above 0.30. In fact, all factor loadings in the different versions were above 0.45. The range of loadings was 0.45–0.81 for the one factor HSCL-10, 0.50–0.83 for the one factor HSCL-5, 0.47–0.82 for the two correlated factor HSCL-10, and 0.51–0.85 for the two correlated factor HSCL-5. Item 17 “Feeling blue” was the item with the highest factor loadings in all the models analyzed. When analyzing the models with two correlated factors, a strong correlation between the two-factors of depression and anxiety was observed for both the HSCL-10 and the HSCL-5.The goodness-of-fit indices in the studied factor models can be consulted in Of the 767 participants who completed the HSCL-25, 736 also took part in the CIDI interview (96.0%). Depression prevalence varied depending on the questionnaire employed . PrevaleThe AUC between the HSCL-10 and the CIDI was 0.877 (CI95% 0.836–0.919). In the gender analysis it was greater in men with an AUC of 0.943 (CI95% 0.897–0.989) compared to women who had an AUC of 0.825 (CI95% 0.765–0.886). The ROC curve is depicted in The AUC between the HSCL-5 and the CIDI was 0.853 (CI95% 0.812–0.894). In the gender analysis it was greater in men with an AUC of 0.918 (CI95% 0.859–0.977) than in women who had an AUC of 0.795 (CI95% 0.734–0.855). The ROC curve is depicted in The optimum cutoff points for the study population were calculated with the Youden Index. For the HSCL-10 it was 1.90 for the total population and for both genders. For the HSCL-5 the optimum cutoff point was 1.80 for the total population and for women, and 2.00 for men. A table including sensitivity, specificity, positive, and negative predictive values using optimal cutoff points is available in the This study is the first to analyze the psychometric properties and diagnostic accuracy of the Spanish versions of the HSCL-10 and HSCL-5, the results obtained now allow the use of both scales as screening tools for depression in the PC setting in Spain. The results demonstrated that in the Spanish PC population, the HSCL-10 presents high reliability and validity. The HSCL-5 also showed acceptable psychometric properties although slightly worse than the HSCL-10. Both scales showed adequate sensitivity and specificity when compared to the semistructured clinical interview CIDI conducted by trained professionals. The optimal cutoff points obtained were very close to those proposed by other authors.The PC setting is ideal for the detection, diagnosis, and investigation of chronic, highly prevalent disorders as it allows an early study of such pathologies . With reThere are many questionnaires that can be employed as screening tools to detect depression at all its stages –52, someWith respect to reliability, Cronbach’s Alpha results of 0.84 and 0.77 were obtained for the HSCL-10 and HSCL-5, respectively. As both were above 0.7, they are considered acceptable . Such fiIn the full 25-item version, 17 “Feeling blue” was the most consistent . This itWhen analyzing Cronbach’s Alpha without the item in the short versions, it was observed that 25 “Sleep disturbance” worsened HSCL-10 reliability, that is to say, by eliminating this item reliability improved. Such a finding concurs with that reported by Kleppang et al. who employed the 10-item version with adolescents in Norway . All the participants gave their written informed consent, and the trial was conducted in accordance with the ethical standards set out in the Declaration of Helsinki. Data were anonymously processed and were used only for the study objectives. Personal data confidentiality was ensured as set out in applicable regulations. Access to medical records was gained as indicated in the legislation currently in force. The patients/participants provided their written informed consent to participate in this study.MR-B, MF-S-M, AC, JL, and PN: conceptualization. EM and IG-G: data collection. MR-B, MF-S-M, and AC: formal analysis. MR-B, MF-S-M, and EP-R: writing—original draft preparation. All authors have reviewed and agreed to the published version of the manuscript.